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Loss of the Arabidopsis thaliana P₄-ATPase ALA3 reduces adaptability to temperature stresses and impairs vegetative, pollen, and ovule development.

McDowell SC, López-Marqués RL, Poulsen LR, Palmgren MG, Harper JF - PLoS ONE (2013)

Bottom Line: We also demonstrate that ala3 mutants have reduced fecundity resulting from a combination of decreased ovule production and pollen tube growth defects.In-vitro pollen tube growth assays showed that ala3 pollen germinated ∼2 h slower than wild-type and had approximately 2-fold reductions in both maximal growth rate and overall length.Together, these results support a model in which ALA3 functions to modify endomembranes in multiple cell types, enabling structural changes, or signaling functions that are critical in plants for normal development and adaptation to varied growth environments.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, University of Nevada, Reno, Nevada, United States of America.

ABSTRACT
Members of the P4 subfamily of P-type ATPases are thought to help create asymmetry in lipid bilayers by flipping specific lipids between the leaflets of a membrane. This asymmetry is believed to be central to the formation of vesicles in the secretory and endocytic pathways. In Arabidopsis thaliana, a P4-ATPase associated with the trans-Golgi network (ALA3) was previously reported to be important for vegetative growth and reproductive success. Here we show that multiple phenotypes for ala3 knockouts are sensitive to growth conditions. For example, ala3 rosette size was observed to be dependent upon both temperature and soil, and varied between 40% and 80% that of wild-type under different conditions. We also demonstrate that ala3 mutants have reduced fecundity resulting from a combination of decreased ovule production and pollen tube growth defects. In-vitro pollen tube growth assays showed that ala3 pollen germinated ∼2 h slower than wild-type and had approximately 2-fold reductions in both maximal growth rate and overall length. In genetic crosses under conditions of hot days and cold nights, pollen fitness was reduced by at least 90-fold; from ∼18% transmission efficiency (unstressed) to less than 0.2% (stressed). Together, these results support a model in which ALA3 functions to modify endomembranes in multiple cell types, enabling structural changes, or signaling functions that are critical in plants for normal development and adaptation to varied growth environments.

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The lipid composition of ala3 pollen is similar to wild-type.Lipid concentrations were measured using tandem mass spectrometry (MS/MS) that detected 11 different head-groups and quantified the acyl carbons and double bonds within the corresponding acyl side chain(s). Concentrations are expressed as a percentage of the total lipid detected for a specific sample and are represented as mean ± SE. Pollen was collected from independent groups (n = 4 for WT and n = 3 for ala3-4) of ∼75 plants each, grown in separate flats, at the same time, in the same growth chamber, under standard (SMB-238 soil, 24°C) conditions. (A) Concentrations of lipid head-groups. Higher-concentration head-groups appear on the left and lower-concentration head-groups appear on the right. (B) Unsaturation in acyl side chain(s). Unsaturation in diacyl lipids (2 acyl chains) appears on the left and unsaturation in lysophospholipids (1 acyl chain) appears on the right. No statistically significant differences between ala3–4 and wild-type were observed (p>0.05, Welch’s t-test) either in terms of head-group concentration or unsaturation. Abbreviations: MGDG, monogalactosyldiacylglycerol; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PA, phosphatidic acid; DGDG, digalactosyldiacylglycerol; PG, phosphatidylglycerol; LPG, lysophosphatidylglycerol; LPC, lysophosphatidylcholine; LPE, lysophosphatidylethanolamine; PS, phosphatidylserine.
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pone-0062577-g007: The lipid composition of ala3 pollen is similar to wild-type.Lipid concentrations were measured using tandem mass spectrometry (MS/MS) that detected 11 different head-groups and quantified the acyl carbons and double bonds within the corresponding acyl side chain(s). Concentrations are expressed as a percentage of the total lipid detected for a specific sample and are represented as mean ± SE. Pollen was collected from independent groups (n = 4 for WT and n = 3 for ala3-4) of ∼75 plants each, grown in separate flats, at the same time, in the same growth chamber, under standard (SMB-238 soil, 24°C) conditions. (A) Concentrations of lipid head-groups. Higher-concentration head-groups appear on the left and lower-concentration head-groups appear on the right. (B) Unsaturation in acyl side chain(s). Unsaturation in diacyl lipids (2 acyl chains) appears on the left and unsaturation in lysophospholipids (1 acyl chain) appears on the right. No statistically significant differences between ala3–4 and wild-type were observed (p>0.05, Welch’s t-test) either in terms of head-group concentration or unsaturation. Abbreviations: MGDG, monogalactosyldiacylglycerol; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PA, phosphatidic acid; DGDG, digalactosyldiacylglycerol; PG, phosphatidylglycerol; LPG, lysophosphatidylglycerol; LPC, lysophosphatidylcholine; LPE, lysophosphatidylethanolamine; PS, phosphatidylserine.

Mentions: To evaluate whether lipid composition is altered in ala3 pollen, the concentrations of 144 lipids were measured in wild-type and ala3 pollen grains using tandem mass spectrometry (MS/MS) (Figure 7, File S1). The MS/MS analysis detected polar lipids from 11 different head-groups (MGDG, PC, PE, PI, PA, DGDG, PG, LPG, LPC, LPE and PS) and quantified the acyl carbons and double bonds within the corresponding acyl side chain(s). We chose to examine pollen grains because expression profiling data suggests that ALA3 is preferentially expressed in mature pollen grains and growing pollen tubes (Figure S4) and because the fitness of ala3 pollen was observed to be temperature-dependent (Table 1). Furthermore, pollen grains could be easily harvested as a pure cell type, minimizing the complications of analyzing tissues made up of different cell types at different developmental stages or physiological states. No differences between ala3 and wild-type pollen were observed in the concentrations of different head-groups (Figure 7a), or in the amount of double bonds (i.e., unsaturation) within acyl side chains (Figure 7b). These results provide evidence that the concentrations of major membrane-associated lipids in ala3 pollen are not detectably different from wild-type under standard growth conditions.


Loss of the Arabidopsis thaliana P₄-ATPase ALA3 reduces adaptability to temperature stresses and impairs vegetative, pollen, and ovule development.

McDowell SC, López-Marqués RL, Poulsen LR, Palmgren MG, Harper JF - PLoS ONE (2013)

The lipid composition of ala3 pollen is similar to wild-type.Lipid concentrations were measured using tandem mass spectrometry (MS/MS) that detected 11 different head-groups and quantified the acyl carbons and double bonds within the corresponding acyl side chain(s). Concentrations are expressed as a percentage of the total lipid detected for a specific sample and are represented as mean ± SE. Pollen was collected from independent groups (n = 4 for WT and n = 3 for ala3-4) of ∼75 plants each, grown in separate flats, at the same time, in the same growth chamber, under standard (SMB-238 soil, 24°C) conditions. (A) Concentrations of lipid head-groups. Higher-concentration head-groups appear on the left and lower-concentration head-groups appear on the right. (B) Unsaturation in acyl side chain(s). Unsaturation in diacyl lipids (2 acyl chains) appears on the left and unsaturation in lysophospholipids (1 acyl chain) appears on the right. No statistically significant differences between ala3–4 and wild-type were observed (p>0.05, Welch’s t-test) either in terms of head-group concentration or unsaturation. Abbreviations: MGDG, monogalactosyldiacylglycerol; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PA, phosphatidic acid; DGDG, digalactosyldiacylglycerol; PG, phosphatidylglycerol; LPG, lysophosphatidylglycerol; LPC, lysophosphatidylcholine; LPE, lysophosphatidylethanolamine; PS, phosphatidylserine.
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getmorefigures.php?uid=PMC3646830&req=5

pone-0062577-g007: The lipid composition of ala3 pollen is similar to wild-type.Lipid concentrations were measured using tandem mass spectrometry (MS/MS) that detected 11 different head-groups and quantified the acyl carbons and double bonds within the corresponding acyl side chain(s). Concentrations are expressed as a percentage of the total lipid detected for a specific sample and are represented as mean ± SE. Pollen was collected from independent groups (n = 4 for WT and n = 3 for ala3-4) of ∼75 plants each, grown in separate flats, at the same time, in the same growth chamber, under standard (SMB-238 soil, 24°C) conditions. (A) Concentrations of lipid head-groups. Higher-concentration head-groups appear on the left and lower-concentration head-groups appear on the right. (B) Unsaturation in acyl side chain(s). Unsaturation in diacyl lipids (2 acyl chains) appears on the left and unsaturation in lysophospholipids (1 acyl chain) appears on the right. No statistically significant differences between ala3–4 and wild-type were observed (p>0.05, Welch’s t-test) either in terms of head-group concentration or unsaturation. Abbreviations: MGDG, monogalactosyldiacylglycerol; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PA, phosphatidic acid; DGDG, digalactosyldiacylglycerol; PG, phosphatidylglycerol; LPG, lysophosphatidylglycerol; LPC, lysophosphatidylcholine; LPE, lysophosphatidylethanolamine; PS, phosphatidylserine.
Mentions: To evaluate whether lipid composition is altered in ala3 pollen, the concentrations of 144 lipids were measured in wild-type and ala3 pollen grains using tandem mass spectrometry (MS/MS) (Figure 7, File S1). The MS/MS analysis detected polar lipids from 11 different head-groups (MGDG, PC, PE, PI, PA, DGDG, PG, LPG, LPC, LPE and PS) and quantified the acyl carbons and double bonds within the corresponding acyl side chain(s). We chose to examine pollen grains because expression profiling data suggests that ALA3 is preferentially expressed in mature pollen grains and growing pollen tubes (Figure S4) and because the fitness of ala3 pollen was observed to be temperature-dependent (Table 1). Furthermore, pollen grains could be easily harvested as a pure cell type, minimizing the complications of analyzing tissues made up of different cell types at different developmental stages or physiological states. No differences between ala3 and wild-type pollen were observed in the concentrations of different head-groups (Figure 7a), or in the amount of double bonds (i.e., unsaturation) within acyl side chains (Figure 7b). These results provide evidence that the concentrations of major membrane-associated lipids in ala3 pollen are not detectably different from wild-type under standard growth conditions.

Bottom Line: We also demonstrate that ala3 mutants have reduced fecundity resulting from a combination of decreased ovule production and pollen tube growth defects.In-vitro pollen tube growth assays showed that ala3 pollen germinated ∼2 h slower than wild-type and had approximately 2-fold reductions in both maximal growth rate and overall length.Together, these results support a model in which ALA3 functions to modify endomembranes in multiple cell types, enabling structural changes, or signaling functions that are critical in plants for normal development and adaptation to varied growth environments.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, University of Nevada, Reno, Nevada, United States of America.

ABSTRACT
Members of the P4 subfamily of P-type ATPases are thought to help create asymmetry in lipid bilayers by flipping specific lipids between the leaflets of a membrane. This asymmetry is believed to be central to the formation of vesicles in the secretory and endocytic pathways. In Arabidopsis thaliana, a P4-ATPase associated with the trans-Golgi network (ALA3) was previously reported to be important for vegetative growth and reproductive success. Here we show that multiple phenotypes for ala3 knockouts are sensitive to growth conditions. For example, ala3 rosette size was observed to be dependent upon both temperature and soil, and varied between 40% and 80% that of wild-type under different conditions. We also demonstrate that ala3 mutants have reduced fecundity resulting from a combination of decreased ovule production and pollen tube growth defects. In-vitro pollen tube growth assays showed that ala3 pollen germinated ∼2 h slower than wild-type and had approximately 2-fold reductions in both maximal growth rate and overall length. In genetic crosses under conditions of hot days and cold nights, pollen fitness was reduced by at least 90-fold; from ∼18% transmission efficiency (unstressed) to less than 0.2% (stressed). Together, these results support a model in which ALA3 functions to modify endomembranes in multiple cell types, enabling structural changes, or signaling functions that are critical in plants for normal development and adaptation to varied growth environments.

Show MeSH
Related in: MedlinePlus